| In recent years,the rates of restoration of spontaneous circulation(ROSC)have gradual advanced,but neurons lack the capacity of regeneration,and are also sensitive to hypoxic/ischemic stress,most patients following ROSC have irreversible neurological injury,result in neurological sequelae such as aphasia,paralysis,vegetable state or even death.Despite comprehensive measures such as mild hypothermia,medicine are adopted to reduce neurological injury,survival rates among patients with cardiac arrest(CA)are still low.In china,only about 1% patients survival to discharge from hospital with good neurological outcome.Reducing neuronal damage and promoting neurological recovery remain to be a critical problem in modern medicine.A new therapy for neurological injury following CA was attempted in the present research.Mesenchymal stem cells(MSCs)have high self-renewal capacity and multilineage differentiation potential.In vitro,MSCs can be repeatedly passaged,expanded in large scale,and differentiate into adipocyte,osteoblasts,cartilage and neuron.Adipose derived stem cells(ADSCs)are MSCs derived from adipose tissue,with unique advantages such as abundant resource in adipose tissue,easiness to acquire by less invasion methods and stability capacity of proliferation and differentiation,which don't decrease with aging.Current researches have demonstrated ADSCs had good neurological outcome in rats with the middle cerebral artery occlusion(MCAO).It is proved that ADSCs can reduce the size of cerebral infarction,alleviate focal neurological injury and promote the recovery of neurological function.Global brain hypoxic injury following CA is different in pathogeny,pathology and clinical features,compared with MCAO,a focal cerebral hypoxic model.Therapeutic mechanism and effect of ADSCs therapy in hypoxic encephalopathy following CA need further verification.The purpose of this study is to evaluate the therapeutic effect and mechanisms of xenotransplantation human ADSCs in rat with hypoxic encephalopathy following CA,and provide theoretical basis for clinic.The main contents include culture and expansion of human ADSCs,identification from cell morphology,cell phenotype and multilineage differentiation ability,preparation of rats cardiac arrest model induced by asphyxia,therapeutic effect of human ADSCs in hypoxic encephalopathy following CA,the expression of brain derived neurotrophic factor(BDNF)and IL-6 in hippocampal tissue.Part ? Culture and identification of ADSCsObjective To establish method for isolation,culture and proliferation of human ADSCs,identify properties of MSCs from cell morphology,cell phenotype and multilineage differentiation ability(osteogenic differentiation,chondrogenic differentiation,adipogenic differentiation),and establish basis for the next experiment.Materials and Methods Adipose tissue was obtained from healthy volunteer by liposuction,and was minced,rinsed repeatedly with PBS solutions,digested with type I and II collagenase digestion under sterile conditions.Stromal vascular fraction(SVF)were cultured on culture media(DMEM/F12 with 10% fetal bovine serum)in an incubator with 37?,5%CO2,and purified to ADSCs.The culture media was changed every 3days.ADSCs were passaged when the cell fusion was over 80% of the flask.Cell morphology was observed during cell culture process.The cell phenotype(CD73,CD90,CD105,CD34,CD45,CD11 b,CD19,HLA-DR)of ADSCs grown for 3 passages were detected using flow cytometry method.Osteogenic differentiation was induced by osteogenic differentiation kit,and detected through alizarin red staining and NBT-BCIP staining at 28 days.Chondrogenic differentiation was induced by chondrogenic differentiation kit,and detected through alcian blue staining at 7days.Adipogenic differentiation was induced by adipogenic differentiation kit,and detected through Oil red O staining at 14 days.Result Primary cells adhered to the dish bottom after 24 huors,which displayed fibroblast-like,spindle,or triangle shaped morphology.Cells began to proliferate and gradually grew to form small colonies.ADSCs were densely distributed,had uniform spindle morphology,and grew in a vortex-like manner.After 6~8days,cell fusion was over 80%.Proliferation capacity was stable in passage cells.The percentage of cell surface marker CD73 positive cells in ADSCs was 99.99%,respectively,of CD90 99.99%,respectively,and of CD105 96.88%,but CD34,CD45,CD11 b,CD19,HLA-DR were detected to be negative.After osteogenic differentiation,cells were vigorous growth,displayed short spindle or irregular shaped morphology,mineralization nodules were observed at 7days.At 28 days mineralization nodules were stained as red spots by alizarin red,which showed red bone-like tissue structures,and alkaline phosphatase(ALP)in osteoclasts cytoplasm as stained as blue precipitate by NBT-BCIP staining.After chondrogenic differentiation,the formation of round or oval nodules were observed under the microscope,cells were stained blue by alcian blue at 7days.After adipogenic differentiation,cells displayed round shaped morphology,lipid droplets were observed in kytoplasm,with positive oil red O staining at 14 days.Conclusion ADSCs derived from human adipose tissue had stability proliferation capacity,showed positive expression of CD73,CD90,CD105,and negative expression of CD34,CD45,CD11 b,CD19 and HLA-DR.ADSCs can differentiate into osteoblasts,chondrocytes and adipose cells.Part ? Therapeutic effect and protection mechanism of human ADSCs in hypoxic encephalopathy following CAObjective To observe changes about neurological function,neurons apoptosis,serum S100? after transplantation of human ADSCs in rat with hypoxic encephalopathy following CA,and investigate the expression of BDNF and IL-6 in hippocampal tissue.Materials and Methods 54 rats were randomly divided into 3 groups: sham group,Cardiac arrest group,and ADSCs group.Rats in sham group only went through surgical procedures,without CA and CPR.Rats in CA group went through CA and CPR and received 1mL PBS buffer by intravenous injection at one hour after restoration of spontaneous circulation(ROSC),while rats in ADSCs group went through CA and CPR and received 1mL ADSCs(5×106 cells)by intravenous injection.At 24,72,and 168 hours after operation,6 rats in each group were randomly selected and euthanized.Hypoxic brain damage was evaluated using Neurological Deficit Scale Score(NDS),serum s100-? was detected by ELISA,and apoptosis rates of hippocampal neural were detected by TUNEL staining,protein levels of BDNF and IL-6 in the hippocampus were detected by western blot.Result 1.Weight,mean arterial pressure and heart rate of rats before CA were collected,there were no significant difference among 3 groups(P>0.05).Duration of asphyxia,duration of CPR and MAP 1hour after ROSC showed no significant difference among 3 groups(P>0.05).2.NDS score of sham group was significantly higher than ADSCs group and CA group at 24 hours,72 hours,168 hours after ROSC(P<0.05),and NDS score of ADSCs group was increased compared with CA group at each time point(P<0.05).3.Hippocampal tissue in sham group was determined to be morphologically normal.In CA group,necrosis of pyramidal cells and proliferation of glial cells were observed in hippocampal CA1 region.ADSCs group also showed necrosis of pyramidal cells,however numbers of necrotic cells were significantly lower in ADSCs group,as compared with CA group.4.At each time point,rats in CA group and ADSCs group showed a large number of TUNEL positive cells after ROSC,which were significantly higher than sham group(P<0.05),and ratio of hippocampal neuronal apoptosis was significantly decreased in ADSCs group,as compared with CA group(P<0.05).5.At each time point,S100? levels in CA group and ADSCs group were significantly higher than in sham group(P<0.01),while levels of S100? were significantly decreased in ADSCs group compared with CA group(P<0.05).6.Compared with IL-6 in sham group,IL-6 levels of hippocampus in CA group and ADSCs group were increased at each time point(P<0.01).At 24 hours and 72 hours after CPR,IL-6 level in ADSCs group was significantly higher than in CA group(P<0.01),while at 168 hours after CPR,IL-6 level showed no significant difference between CA group and ADSCs group(P>0.05).7.At 24 hours and 72 hours after ROSC,BDNF protein levels in CA group and ADSCs group were significantly higher than in sham group(P<0.01),while BDNF protein level were significantly increased in ADSCs group compared with CA group(P<0.01).At 168 hours after ROSC,BDNF protein levels showed no significant difference among 3 groups(P>0.05).Conclusion CA cause severe neurological dysfunction and neuronal apoptosis,ADSCs transplantation can reduce neurological injury and improve the prognosis of neurological function.The neuroprotective effect is related with up-regulated expression of BDNF and IL-6. |
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